MicroDiagnostics

Tabbed contents

Main research areas include liquid biopsy-based procedures, point-of-care tests for the detection of infections, cancer or neurodegenerative diseases aiming to provide a safer and faster answer to the patient.

The system developments include the specific preparation of samples for the isolation of target molecules from complex matrices, the development of molecular detection methods as well as their integration and evaluation.

In addition, the applications of novel chemo- and biosensors as well as a platform for smartphone-based diagnostics are being developed.

HyFly – Non-invasive diagnostics for infectious diseases

Modern air traffic is not just useful for the fast transport of passengers or goods. Even infectious agents travel by planes over long distances within a few hours as unwanted passengers. Infectious diseases such as influenza or SARS, which can develop into pandemics, are nowadays spreading rapidly and much faster than years ago. Steps to effectively control and prevent chains of infection in the field of modern mobility have not yet been effectively established worldwide. Initial approaches using simple questionnaires or non-contact temperature measurement, however, have remained largely ineffective. The project "HyFly", funded by the German Federal Ministry of Education and Research (BMBF) within the InfectControl 2020 initiative, addresses the sensitive issue of passenger control. Together with partners, a non-invasive method based on ion mobility spectrometry (IMS) is being worked on. Applying this strategy, infected persons should be identified within a few minutes via components of their breathing air. IMS is already routinely used worldwide to detect drugs or explosive remnants in airports providing an established infrastructure. So-called volatile organic substances that are metabolic products of microorganisms are detected. The focus is on bacterial pathogens, which, according to information from the German Robert Koch Institute, have a high relevance for aviation. Initial results show that the method has great potential for discriminating between different pathogens. In addition to system development, a study is underway to identify microorganisms at various international airports to determine the cleaning efficiency and impact of antimicrobial coatings.

Sample Preparation

Sample preparation is a crucial aspect in many areas of bioanalytical research, especially in the analysis of the crude complex samples and/or rare targets. Modern laboratories exploit very sensitive methods of detection including molecular diagnostics; however, their performance strongly depends on the quality of the sample. Pre-analytical processing must prepare the specimen for the most effective detection of the target. It includes the purification of the analyte, its pre-concentration, as well as the parallel removal of the compounds which may affect the analysis. The preservation of samples which prevents the degradation of the target is also a task for this field of applied analytics. The main aim of sample preparation is to ensure the precision of the subsequent analysis. None of the sample preparation approaches are universal: they must take into account methods and equipment for the downstream processing of the specimen, concentration and nature of the analyte, volume of the sample and many other factors.

Nowadays bioanalytical research actively pursues integrating complex assays on automated platforms including lab-on-chip devices. This trend often lacks intelligent solutions for the pre-analytical steps. The aim of our working group is to support this particular field by developing of the most suitable sample preparation approaches for specific needs.

The group supports researchers and industrial partners with customized solutions, evaluates capacities of existing methods and develops novel strategies for effective pre-analytical processing.

Parodontitis chip

Taking samples for the rapid analysis of periodontalpathogenic agents using the parodontitis chip.

Periodontitis is an inflammatory disease of the gums that, if left untreated, can lead to tooth loss. In Germany alone it is predicted that nearly 12 million people are affected by periodontitis. The main trigger for periodontal disease is bacterial plaque which can lead to a reduction of the dental bone tissue. The postulated systematic relationship between periodontal disease caused by bacterial pathogens and cardiovascular damage has been studied extensively. It can result in particularly serious diseases such as heart attacks and strokes.

The parodontitis chip project is aimed at developing a fully integrated diagnostics platform both for the fast processing and the subsequent analysis of periodontal pathogens in complex samples. This innovative technology consists of a compact microfluidic card and a combined purification module. Steps such as isolating pathogenic nucleic acids, selectively amplifying DNA sequences, and their specific detection are integrated to establish an easy-to-use setup for the end-user.

The lab-on-a-chip device will allow the detection and characterization of 11 bacteria relevant to the pathogenesis of periodontitis in a parallel format. In addition, the establishment of a simple detection unit will allow the monitoring of reaction kinetics. Therefore a quantification of the pathogen, as well as a determination of the total bacterial count can be realized.

The parodontitis chip project will allow for the creation of a simple molecular diagnostic test platform that can easily be adapted to various problems in the field of medical, environmental, or food analysis. Simplified lab-on-chip devices having an extremely simple structure and non-contact detection units provide significant time and cost savings for the user.

Development of diagnostic test strip systems

The significance of easy-to-use test strip systems for the rapid detection of clinically relevant parameters or for quality assurance of food products is increasing not only in developing countries. We develop a simple diagnostic platform that is particularly suitable for nucleic acid-based formats. As a reference assay, pathogens are diagnosed in human samples.

TBSearch project

Tuberculosis is an infectious disease, which is caused by Mycobacterium tuberculosis. According to a 2013 report of WHO (World Health Organisation), Tuberculosis ranks as the second leading cause of death from an infectious disease worldwide, after the human immunodeficiency virus (HIV).

An early and reliable diagnosis is crucial. Our unit is currently developing in cooperation with the McMaster University in Hamilton (Canada) a detection system which is rapid, simple, and cost-efficient. The nucleic acid-based test system integrates all steps from pathogen isolation to hybridization of nucleic acid on pathogen-specific probes.

Rapid on-site diagnosis of infectious diseases based on a lab-on-a-chip system

Controlling magnetic nanoparticles through a microfluidic chip – produced by the rapid prototyping method – with the aid of magnets that are arranged above and below the microfluidic chip.

A reliable diagnosis of complex and life-threatening infectious diseases (e.g. sepsis) is currently only possible using elaborate and time-consuming methods involving an analysis laboratory and qualified specialists. The Unit is developing an innovative system for rapid, easy-to-conduct and inexpensive on-site infection diagnostics.

The system is based on magnetic particles in the micrometer scale that can be functionalized according to their respective application to act as carriers for antibodies and disease-associated DNA sequences. These magnetic particles are employed on a disposable object in the approximate shape of a check card. In an on-site examination a sample is obtained from the patient, such as blood, saliva or urine, which is then incorporated into the lab-on-a-chip system. After lysis of the target cells, the magnetic particles bind to the respective target molecules in the sample and are transported via magnetic forces through different reaction tubes in a fully automated manner. At the end of the process chain the diagnosis is performed using a highly sensitive magnet sensor system.

The project is funded by the Federal Ministry for Education and Research (BMBF, Bundesministerium für Bildung und Forschung) and coordinated by Magna Diagnostics GmbH, a spin-off company of the Fraunhofer IZI.

Production and characterization of DNA-binding proteins

A plurality of different proteins exhibit specific DNA binding properties that are associated with targeted cellular functionalities. We utilize zinc finger proteins that bind specific nucleic acid sequences and are capable of capturing double-stranded DNA from sample solutions when used as immobilized components of a protein array.

Furthermore, we focus on functional proteins that are capable of isolating pathogens from an excess of human DNA. These proteins are intended to be employed in novel purification methods.

NoDope – detection of doping misuse by athletes

Athletes have been trying to improve their performance through illegal means for decades. They use specific products that can enhance endurance, muscle growth and strength or improve recovery after extensive trainings/contests.

Within this project, a diagnostic device is developed to detect different doping products in blood samples. A complete integration of sample preparation and bioassay will be designed and developed. Furthermore, the power of this device will be the detection of multiple doping products in one assay based on surface plasmon resonance (SPR) technology.

Reference project: System validation for ApoStream™ technology

Circulating tumor cells (CTCs) are cancer cells shed from a primary tumor and circulated in the peripheral blood. Detection and isolation of CTCs can be used for diagnostics purposes and e.g. personalized medicines. Circulation tumor cells (CTCs) differ from peripheral blood mononuclear cells (PBMCs) in many ways. Detecting CTCs in blood samples is however a challenge, since CTCs comprise a very rare population among red blood cells and leukocytes.

The ApoStream™ technology from ApoCell can detect and enrich CTCs from blood with the use of a method called dielectrophoresis (DEP). This device is still in development and in order to validate the system, surrogates for CTCs and PBMCs need to be developed. Both biological as non-biological surrogates are designed, produced, screened and optimized.

Reporter gene assays offer a wide range of applicabilities in modern research. In the first place, they serve for characterizing regulatory elements (promoter regions) or modulators (transcription factors) on the genomic level. In particular, luciferase-based reporter gene assays are established in current biomedical and pharmacological research. Due to their extremely low detection limit they have prevailed over fluorescence-based reporter genes. Basically, the regulatory genetic element to be investigated is cloned upstream of a luciferase gene. The reporter gene construct can be introduced into a selected cell system by means of conventional transfection methods. The biological activity of the cloned genetic element can now be characterized in a time-dependent manner. The Fraunhofer IZI offers a complete dual-luciferase system that is suitable for the investigation of genetic regulatory elements from mammalian genomes.

Method

The method comprises three steps:

cloning and sequencing the target sequence into the reporter construct

Reference project

In a cooperation project with the department of General Biochemistry of the University of Leipzig we succeeded in investigating the activity of the human GLO1 promoter in different carcinoma cell lines. At present, the influence of hypoxia on promoter activity is being investigated.

Selected applications

The system is used for the functional characterization of any conceivable regulatory element from mammalian genomes. Moreover, the system offers a way of examining exogenous factors in combination with normal or altered culture conditions.

RNAi is a conserved mechanism that regulates gene expression on the post-transcriptional level. In eukaryotes, double-stranded RNA (dsRNA) is processed to form short, small interfering RNAs (siRNA) which results in the degradation of the complementary mRNA. Finally, this leads to the effective down-regulation of the specific protein. These characteristic features result in the application of RNAi in cell cultures and animal models. The specific suppression of gene expression and protein activity modulates the pharmacological inhibition of the target protein and is thus an effective tool in proof-of-principle experiments and the identification and validation of antitumor agents.

For providing a novel cancer therapy, the reference project aims at the specific and gradual down-regulation of an already identified target protein known as glyoxalase I (GLO I) by means of RNAi. Moreover, the system shall provide a way of re-up-regulating GLO I in order to recreate the initial phenotype.

In tumors there is a strong correlation between signal transduction pathways and fundamental metabolic pathways, such as glycolysis and the pentose phosphate cycle. Many tumor-promoting mechanisms have an immediate effect on glycolysis, on the cellular response to oxygen and on the ability of tumors to recruit new vessels for their own nourishment. Since Warburg it has been known that tumors consistently utilize anaerobic pathways to produce ATP by conversion of glucose. One cytotoxic byproduct of glycolysis is methylglyoxal (MGO). The reactive MGO binds to proteins and nucleic acids in high concentrations, thereby inducing apoptosis. All organisms have a glyoxalase enzyme system (GLO I / GLO II) to prevent cell damage caused by high MGO concentrations. In particular in tumor cells, this system is up-regulated in order to minimize the paracatalytically generated high MGO concentrations. The inhibition of glyoxalases could therefore play an important role in cancer therapy. To date, the characterization of the glyoxalases in malignant tumors only referred to histochemical analyses which show an overexpression in tumors. By means of the RNAi model, specific GLO I inhibitors could be identified in substance libraries.